Human Cu,Zn superoxide dismutase (SOD1) converts superoxide, a toxic byproduct of oxidative phosphorylation, into water and oxygen in all respiring cells. Tragically, mutations at dozens of positions in SOD1 cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. A prevailing hypothesis for the molecular mechanism of the disease involves the aggregation of marginally-soluble forms of SOD1 whose populations are increased by the ALS-inducing mutations. Biophysical analysis of the folding mechanism of this dimeric -barrel protein and quantitative assessment of the perturbations in the populations of monomeric forms induced by mutations, performed during the previous grant period, support this hypothesis. The overall goals of the present proposal are (1) to examine the structural, thermodynamic and kinetic properties of the rate-limiting monomer folding reaction in ALS-variants of SOD1 and (2) to develop and apply fluorescence-based optical methods to monitor directly the subsequent diffusion-limited subunit association reaction and the formation of oligomers during the early stages of the aggregation reaction. A combined denaturant and temperature analysis of the kinetic folding reaction will provide insights into the thermodynamic properties of the transition state ensemble (TSE) controlling the formation of the folded monomeric state. A complementary mutational analysis will highlight the side chains involved in defining the TSE and test the role of hydration in determining the barrier for this extraordinarily slow reaction. Small angle x-ray scattering experiments will probe the size and shape of monomeric ALS variants of SOD1. The resistance of main chain amide hydrogens to exchange with solvent will be monitored by mass spectrometry and NMR spectroscopy to probe for persistent secondary structure in partially-folded states of SOD1 and several ALS variants. The results will be compared with the protection patterns observed in aggregates of the ALS variants. Forster resonance energy transfer and fluorescence correlation spectroscopy will be used to monitor the subunit association reaction and the early stages of the aggregation reaction for several ALS variants labeled with extrinsic chromophores. The results will provide a quantitative framework for describing the effects of ALS-inducing mutations on SOD1 and insights into the mechanism by which they enhance its propensity to aggregate.